A Novel Differential Pulse Voltammetric (DPV) Method for Measuring

Article pubs.acs.org/JAFC

A Novel Differential Pulse Voltammetric (DPV) Method for Measuring the Antioxidant Capacity of Polyphenols-Reducing Cupric Neocuproine Complex Ayşe Nur Tufan, Sefa Baki, Kubilay Gücļ ü, Mustafa Ö zyürek, and Reşat Apak* Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey ABSTRACT: A novel differential pulse voltammetric (DPV) method is presented, using a chromogenic oxidizing reagent, cupric neocuproine complex (Cu(Nc)22+), for the assessment of antioxidant capacity of polyphenolic compounds (i.e., flavonoids, simple phenolic acids, and hydroxycinnamic acids), ascorbic acid, and real samples for the first time. The electrochemical behavior of the Cu(Nc)22+ complex was studied by cyclic voltammetry at a glassy carbon (GC) electrode. The electroanalytical method was based on the reduction of Cu(Nc)22+ to Cu(Nc)2+ by antioxidants and electrochemical detection of the remaining Cu(II)−Nc (unreacted complex), the difference being correlated to antioxidant capacity of the analytes. The calibration curves of individual compounds comprising polyphenolics and vitamin C were constructed, and their response sensitivities and linear concentration ranges were determined. The reagent on the GC electrode retained its reactivity toward antioxidants, and the measured trolox equivalent antioxidant capacity (TEAC) values of various antioxidants suggested that the reactivity of the Cu(II)−Nc reagent is comparable to that of the solution-based spectrophotometric cupric ion reducing antioxidant capacity (CUPRAC) assay. This electroanalytical method better tolerated sample turbidity and provided higher sensitivity (i.e., lower detection limits) in antioxidant determination than the spectrophotometric assay. The proposed method was successfully applied to the measurement of total antioxidant capacity (TAC) in some herbal tea samples such as green tea, sage, marjoram, and alchemilla. Results demonstrated that the proposed voltammetric method has precision and accuracy comparable to those of the spectrophotometric CUPRAC assay. KEYWORDS: total antioxidant capacity, differential pulse voltammetry, polyphenolics, tea samples, CUPRAC method

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INTRODUCTION Measuring the antioxidant activity/capacity levels of food and biological fluids serves multiple purposes such as aiding the diagnosis and treatment of oxidative stress-associated diseases (e.g., muscle and tissue degeneration, heart disease, diabetes, and cancer) in clinical biochemistry, comparison and classification of foods in regard to their antioxidant content, and control of variations within or between foods and nutraceutical products.1 Epidemiological studies on the determination of TAC have shown that a diet rich in food products can reduce the risk of cardiovascular diseases and certain cancers.2,3 The phenolic compounds (i.e., flavonoids, hydroxycinnamic acids, and hydroxybenzoic acids) in complex matrices such as fruits, fruit juices, vegetables, and biological samples are generally determined by using different analytical techniques, such as spectrophotometric, chromatographic, chemiluminescent, and electrochemical methods.4 Recently, electrochemical methods such as cyclic (CV), differential pulse (DPV), and square-wave voltammetry (SWV) have been used for determining natural and synthetic phenolic compounds,4,5 and for predicting their antioxidant capacity in different matrices, because polyphenols acting as reductants are electrochemically active. DPV is one of the most sensitive techniques and has received a great deal of attention in recent years. CV methods have also been described to detect ascorbic acid, citric acid, and sugars in both food products and pharmaceuticals and to consider their influence on the oxygen reduction © 2014 American Chemical Society

process, but this approach is restricted due to the electrochemically inactive nature of these compounds in the potential range of O2 reduction.6 Qualitative assessment of wine phenolics based on reducing strength was also realized by using CV at a GC electrode; although in this approach, the area under curve (AUC), namely, the charge passed to 0.5 V potential during CV recording, is understood as a better estimate of the concentration of polyphenols with low oxidation potentials, quantitation efforts of all reducing compounds may only provide a qualitative picture in a complex mixture such as wine mainly because the magnitude of the response is not identical on a molar basis for dissimilar compounds.7 Although the DPV-based method for measuring drug total antioxidant capacity (TAC) generally seemed to be sufficiently in line with the results of reference methods, its precision was rather poor (RSD > 10%).8 Novak et al.5 used SWV to investigate the electrochemical behavior of major green tea compounds such as epigallocatechin gallate, epigallocatechin, and gallic acid. Magarelli et al.4 developed and validated a sensitive DPV method using a GC electrode for the determination of total phenolic acids in cotton cultivars; however, the authors studied only four polyphenolics (i.e., caffeic, chlorogenic, gallic, and gentisic acids) and presented one anodic peak at approximately 0.4 V that was assigned to the Received: Revised: Accepted: Published: 7111

April 14, 2014 June 29, 2014 July 5, 2014 July 6, 2014 dx.doi.org/10.1021/jf5017797 | J. Agric. Food Chem. 2014, 62, 7111−7117

Journal of Agricultural and Food Chemistry

Article

Teflon closed vessels with an automatic fiber optic temperature control system; dried sample (0.5 g) was extracted with 10 mL of EtOH/H2O (4:1, v/v) mixtures at 80 °C for 3 min after 3 min of temperature balancing time, and the microwave power (0−1500 W) was adjusted automatically according to temperature (5 min). The obtained ethanolic extracts were filtered through a filter paper, then through 0.45 μm PTFE syringe filters (Whatman), and kept at 4 °C until use. All standard solutions of antioxidant polyphenols were initially prepared in absolute EtOH at a concentration of 10 mM and diluted (to 1 mM) using bidistilled water (Millipore Synergy UV, Molsheim, France). Thus, the polyphenol working solutions were finally in 10% EtOH medium, except quercetin, which was finally prepared in 50% EtOH due to the aqueous solubility limitation. All standard solutions were stored at −20 °C prior to analysis. For spectrophotometric measurements, CuCl2 solution (10 mM) and ammonium acetate buffer solution (1 M, pH 7.0) were prepared in bidistilled water, and neocuproine solution (7.5 mM) was prepared in EtOH. For electrochemical measurements, CuCl2 solution (2 mM) and acetic acid/acetate buffer solution (supporting electrolyte, 0.1 M, pH 4.76) were prepared in bidistilled water, and neocuproine solution (4 mM) was prepared in EtOH. DPV−CUPRAC Assay (Proposed Method). DPV was performed using a potentiostat model Reference 600 (Potentiostat/Galvanostat/ ZRA, Gamry) in a three-electrode configuration, employing a GC electrode (Gamry, 3 mm disk, 7 mM OD) as the working electrode, platinum wire as the counter electrode, and Ag/AgCl (∼5 mol/L KCl) as the reference electrode. The GC electrode was polished before electrochemical measurements; that is, the electrode surface was manually polished with an alumina suspension (Baikowski Int. Corp.; 0.05 μm) on polishing cloth for 2 min and rinsed with distilled water; then, the electrode was immersed in EtOH/H2O (1:1, v/v) mixture, transferred to an ultrasonication bath for 3−4 min, and rinsed with ultrapure water. Finally, the electrode was immersed in distilled water and ultrasonication was repeated. The required volume of antioxidant standards and samples (herbal tea extracts) was added by micropipet into a mixture solution of the supporting electrolyte and Cu(II)−Nc chelate in the electrochemical cell. The reaction mixture was left to react for ∼1 min on a stirrer at room temperature. The voltammograms were recorded immediately to minimize adsorption of polyphenols (or other electroactive species) onto the GC electrode surface. DPV conditions were as follows: scan range, from +0.6 to −0.25 V; pulse size, 50 mV; step size, 4 mV; pulse time, 0.1 s; and sample period, 0.5 s. The cyclic voltammograms were recorded on a freshly polished GC electrode by scanning the potential from +0.8 to −0.2 V at a scanning rate of 40 mV/s. The TAC values of herbal teas were determined by DPV experiments, and the results were expressed as millimoles of trolox equivalents per gram of tea (mmol TE/g tea). The standard calibration curves of trolox and other phenolics were obtained by measuring the currents (Ip) at the cathodic reduction peak potential of the Cu(Nc)22+/Cu(Nc)2+ redox couple before and after reaction with antioxidants, taking the difference (ΔIp), and plotting this difference versus concentration of antioxidant standard. Spectrophotometric CUPRAC Assay. The spectrophotometric CUPRAC method described by Apak et al. is based on the reduction of a cupric neocuproine complex (Cu(II)−Nc) by antioxidants to the highly colored cuprous form (Cu(I)−Nc).12 One milliliter of CuCl2, 1 mL of Nc solution, and 1 mL of NH4Ac solution were added to x mL of the analyte, followed by (1.1−x) mL of H2O. The absorbance of the final solution (of 4.1 mL total volume) at 450 nm was read against a reagent blank after 30 min of standing at room temperature. The absorption measurements were recorded in matched quartz cuvettes using a Varian CARY Bio 100 UV−vis spectrophotometer (Mulgrave, Victoria, Australia) having a spectral resolution of ≈1 nm. The calibration curves (absorbance versus concentration graphs) of each antioxidant were constructed under the described conditions, and their TEAC coefficients, found as the ratio of the molar absorptivity of the concerned compound to that of trolox in the CUPRAC assay, were calculated. All spectrophotometric analyses were carried out in triplicate and performed at room temperature.

oxidation of phenolic hydroxyls leading to the formation of Oquinone via semiquinone form. Therefore, it may be argued whether these four easily oxidized phenolic acids are true representatives of “total phenolic acids” having diverse oxidation potentials. There is very limited study about the usage of GC electrodes as electrochemical TAC sensors. Milardović et al. developed an amperometric antioxidant activity method, based on the reduction of 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH•) at the GC electrode; because the cyclic voltammograms of a number of water- and ethanol-soluble common antioxidants gave either irreversible or undefined oxidation peaks, electrochemical reduction of DPPH• was conducted in the presence and absence of antioxidants, but potential selection was critical due to the electrochemical interferences of caffeic acid and trolox.9 The electrochemical ABTS•+ assay is based on measuring catalytic voltammetric currents caused by antioxidants acting as reductant toward the electrochemically generated ABTS2+ (thereby enabling reoxidation of ABTS•+ on the electrode) in edible oils; this method produced rather high blank values in the trolox calibration curve, and trolox addition to sunflower oil matrices did not yield perfectly linear responses.10 It may be argued that novel electrochemical TAC methods should be based on indirect determinations, that is, on the redox behavior of a single electroactive species capable of reacting with antioxidants with a definite stoichiometry and kinetics, because, otherwise, irreversible or undefined peaks of phenolics oxidation may emerge within a range of potentials, making an evaluation of the results obtained with complex samples very difficult and sometimes inaccurate. Although an electrochemical CUPRAC method (based on CV and chronoamperometry) of antioxidant analysis has very recently been published,11 it does not cover a wide range of antioxidant compounds, and there is no DPV method in the literature for the determination of cupric reducing antioxidant capacity of complex samples. Therefore, the aims of this study are to develop a new electrochemical technique using DPV− CUPRAC methodology for pure phenolic antioxidants, ascorbic acid, and their complex matrices such as herbal teas and to compare the results obtained by the proposed assay with those obtained by spectrophotometric CUPRAC assay. Compared to other chromogenic reagents of similar properties (i.e., DPPH• and ABTS•+), the CUPRAC reagent has been shown to be much less dependent on phenolic lipophilicity, steric effects of accessibility, solvent composition, pH, dissolved oxygen, and daylight.12

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MATERIALS AND METHODS

Chemicals. The following chemicals of analytical reagent grade were supplied from the corresponding sources: copper(II) chloride, ammonium acetate, hydrochloric acid, sodium hydroxide, potassium chloride, ascorbic acid, and syringic acid were purchased from Merck Chemicals (Darmstadt, Germany); gallic acid, neocuproine, chlorogenic acid, (−)epicatechin, p-coumaric acid, quercetin, ferulic acid, vanillic acid, trolox, acetic acid, nitric acid, methanol, and absolute ethanol were supplied from Sigma-Aldrich Chemicals (Steinheim, Germany); (−)-catechin and kaempferol were obtained from Fluka (Buchs, Switzerland). Samples and Standards. The herbal tea samples (green tea, marjoram, sage, and alchemilla) were obtained from local markets (Istanbul, Turkey). The fine dried herbal tea samples were extracted with the aid of a microwave-assisted extraction (MAE) system (Milestone ETHOS ONE, Shelton, CT, USA). MAE was utilized in 12 7112

dx.doi.org/10.1021/jf5017797 | J. Agric. Food Chem. 2014, 62, 7111−7117


Article

Statistical Analysis. Statistical analyses were realized using Excel software (Microsoft Office 2002) for calculating the means and the standard errors of the means. Results were expressed within 95% confidence interval as mean ± (t0.05s/n1/2), where t0.05 is the Student’s table t value for 4 degrees of freedom and s is the standard deviation of n = 5 measurements. Using SPSS software for Windows (version 13), the data were interpreted by two-way analysis of variance (ANOVA).13

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RESULTS AND DISCUSSION Electrochemical Behavior of Cu(Nc)22+ Reagent. The cyclic voltammogram of Cu(Nc)22+ reagent (prepared in 1:2 molar ratio of metal to ligand, i.e., 2 mL of 2 mM Cu(II) + 2 mL of 4 mM Nc) in 6 mL of 0.1 M acetic acid/acetate buffer solution (supporting electrolyte, pH 4.76) at a glassy carbon electrode shows the existence of Cu(Nc)22+/Cu(Nc)2+ redox couple (Figure 1). An oxidation (anodic) peak appeared at

Figure 2. Differential pulse voltammograms of (a) acetic acid/acetate buffer solution (0.1 M, pH 4.76) (supporting electrolyte), (b) acetic acid/acetate buffer solution + 0.4 mM Cu(II), (c) acetic acid/acetate buffer solution + 0.8 mM Nc, (d) acetic acid/acetate buffer solution + Cu(II) + Nc, (e) acetic acid/acetate buffer solution + Cu(II) + Nc + 0.08 mM TR, and (f) acetic acid/acetate buffer solution + 0.08 mM TR. Scans range from +0.7 to −0.25 V; pulse size, 50 mV; step size, 4 mV; pulse time, 0.1 s, and sample period, 0.5 s.

cathodic voltammetric currents decrease further, because the Cu(Nc)22+ complex in the media is reduced to the Cu(Nc)2+ form in proportion to trolox. Thus, the initial amount of Cu(Nc)22+ cannot be maintained upon trolox addition, causing a pronounced decrease in cathodic currents (Figure 3A,B). On the other hand, with increasing trolox concentrations, more Cu(Nc)2+ will be produced, and its electrochemical oxidation (anodic peak) currents at approximately 0.5 V show a slight increase (Figure 3A). The decrease in cathodic peak current upon trolox addition is taken as a measure of trolox concentration (Figure 3C). The dependence of ΔIp on trolox concentration (CTR) is presented in Figure 3C, where ΔIp is defined as the difference between the measured DPV peak currents in the absence of trolox (Ip,0) and in the presence of corresponding trolox concentrations (Ip,x), that is, ΔIp = Ip,0 − Ip,x. The calibration equation corresponding to the regression line as ΔIp versus Ctrolox using the DPV method was ΔIp/μA = 46.66CTR/mmol L−1 + 0.019. This equation was also used for TAC determination of herbal teas, reported as trolox equivalents. The linear concentration range for trolox in the proposed method was 0.001−0.005 mM (correlation coefficient r = 0.9995, N = 5), within which the relative standard deviation (RSD) was 3.1%. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the equations LOD = 3sbl/m and LOQ = 10sbl/m, respectively, where sbl is the standard deviation of a blank and m is the slope of the calibration line. The LOD and LOQ for trolox were found as 0.18 and 0.61 μM, respectively. Determination of Polyphenols by Voltammetric and Spectrophotometric Methods. The chromogenic oxidizing reagent of the spectrophotometric CUPRAC method, that is, (Cu(II)−Nc) chelate, reacts with n-electron reductant antioxidants (AO) in the following reaction:12,15

Figure 1. Cyclic voltammogram of Cu(Nc)22+ reagent (scan rate = 40 mV/s): (a) acetic acid/acetate buffer solution (0.1 M, pH 4.76) (supporting electrolyte); (b) acetic acid/acetate buffer solution + 0.4 mM Cu(II) + 0.8 mM Nc. CV scan was made negatively from +0.8 to −0.2 V (one cycle) and at a rate of 40 mV/s.

approximately 0.5 V and a reduction (cathodic) peak at approximately 0.41 V. Compared to the electrochemical CUPRAC method of Cárdenas et al. carried out in pH 7 ammonium acetate buffer,11 it can be clearly seen from the voltammogram in Figure 1 that pH 4.76 acetate buffer produced better electrochemical reversibility. Figure 2 shows the DPVs of copper(II), cupric−neocuproine complex, and trolox in acetic acid/acetate buffer medium and under identical conditions. It is apparent from this figure that the presence of 2 equiv of neocuproine per 1 equiv of copper(II) leads to a substantial change (of about 0.43 V) in the potential of the Cu(II)/Cu(I) redox couple due to the selective stabilization of tetrahedral cuprous state by neocuproine (i.e., the logarithmic stability constants of the concerned copper chelates, Cu(Nc)22+ and Cu(Nc)2+, have been reported as 12 and 19, respectively).14 The reduction potential of the cupric−neocuproine complex was found at approximately 0.41 V with DPV. The peak current of cupric− neocuproine (Figure 2d) decreased as a result of reaction with trolox (Figure 2e) without a significant shift in reduction potential. Electrochemistry of Cu(Nc)22+ Reagent in the Presence of Trolox. Concentrations of trolox >0.001 mM led to a corresponding decrease of the cathodic peak currents (see Figure 3A,B). When trolox is added in the electrochemical cell containing Cu(Nc)22+, it is easily oxidized to the quinone form, whereas Cu(II)−Nc is reduced to the highly colored Cu(I)− Nc chelate. As the concentration of trolox increases, the

nCu(Nc)2 2 + + n‐electron reductant (AO) → nCu(Nc)2+ + n‐electron oxidized product + nH+ (1)

In this reaction, the reactive Ar−OH groups of polyphenolic antioxidants are oxidized to the corresponding quinones (e.g., 7113



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Antioxidant capacities of 12 antioxidant compounds, namely, hydroxybenzoic acids (vanillic acid, gallic acid, syringic acid), hydroxycinnamic acids (p-coumaric acid, ferulic acid, chlorogenic acid), flavonols (kaempferol and quercetin), flavon-3-ols (epicatechin and catechin), and others (trolox and ascorbic acid), were electrochemically determined. Table 1 summarizes the related linear equations, correlation coefficients, linear ranges, and LOD and LOQ values of the antioxidant compounds tested by the DPV−CUPRAC method (for comparison, the corresponding values found by the conventional spectrophotometric−CUPRAC method are also listed in Table 1). The electrochemical determination of the antioxidant capacity of these phenolic groups (except for pcoumaric and ferulic acids) was performed under the same experimental conditions used for quantifying trolox (Table 1). A linear response of the peak current as a function of concentration was observed for phenolic compounds over the concentration ranges studied. The LOD values of the phenolics found with the DPV− CUPRAC method were lower than those found with spectrophotometric−CUPRAC. The LOD (0.18 μM) found by the recommended DPV method was much lower than that of the electrochemical CUPRAC method of Cárdenas et al.11 It is also noteworthy that the LOD values for CT and CGA found with the proposed DPV procedure (0.15 and 0.24 μM, respectively) are lower than the corresponding LODs of 1.03 and 0.28 μM, reported by Blasco et al., who used a flow injection system with a glassy carbon electrode.17 The voltammetric trolox equivalent antioxidant capacity (TEACvoltammetric, defined as mM trolox equivalents of 1.0 mM antioxidant solution under investigation) was simply calculated by dividing the slope of the linear calibration equations of phenolics under investigation by that of trolox under corresponding conditions (i.e., the slope of trolox was 46.66 μA/mM) using eq 2. TEACvoltammetric =

Figure 3. Cyclic (A) and differential pulse (B) voltammograms of Cu(Nc) 2 2+ reagent, showing the effect of increasing trolox concentration in the range of 0.001−0.005 mM; (C) dependence of ΔIp on trolox concentration. ΔIp is defined as the difference between the measured DPV peak currents in the absence of trolox (Ip,0) and in the presence of corresponding trolox concentrations (Ip,x), that is, ΔIp = Ip,0 − Ip,x. Supporting electrolyte was acetic acid/acetate buffer solution (0.1 M, pH 4.76), and other conditions were the same as in Figure 2.

slopeAO slopeTR

(AO, antioxidant; TR, trolox) (2)

The spectrophotometric trolox equivalent antioxidant capacity (TEACspectrophotometric) values of the phenolics in 10% ethanol were likewise calculated by dividing the molar absorptivity (ε) of each antioxidant by the molar absorptivity of trolox (εtrolox was 15.36 L mmol−1 cm−1 under the specified conditions) using eq 3.

ascorbic acid is 2e-oxidized to dehydroascorbic acid, and trolox is 2e-oxidized to the corresponding quinone), whereas Cu(II)− Nc is reduced to the highly colored Cu(I)−Nc chelate showing maximum absorption at 450 nm. Spectrophotometric CUPRAC is a relatively fast assay, where most of the tested antioxidants reach ≥80% of their maximal molar absorptivities within ≥1 min and do not show a distinct capacity change within 30 min. However, slow-reacting antioxidants (i.e., naringenin, hesperetin) may show a dramatic capacity change within 30 min.12,16 Therefore, the relatively short measurement time of the electrochemical CUPRAC method does not pose major problems in electron-transfer kinetics of most antioxidants. The recommended voltammetric method for TAC assay is based on the electrochemical reduction of Cu(Nc)22+ at the GC electrode polarized at a potential lower than the reduction potential of CuII,I(Nc)2 redox couple (which is about 0.6 V).

TEACspectrophotometric =

εAO εTR

(3)

The TEACvoltammetric and TEACspectrophotometric values were compared (Table 2) to yield compatible results. For the tested phenolic antioxidants, the voltammetric method correlated linearly with spectrophotometric method (r = 0.9791, N = 5) (Figure 4). A similar investigation on the interrelationship of spectrophotometric and DPV10 modification of ABTS/ persulfate assay gave slightly different results for the TEAC coefficients of gallic and ascorbic acids (unreported data), naturally requiring more measurements on antioxidant varieties to reach any conclusion. It should be remembered here that spectrophotometric−CUPRAC measures the absorbance of the Cu(Nc)2+ formed at pH 7.0, whereas voltammetric−CUPRAC measures the reduction current of the remaining Cu(Nc)22+ at pH 4.76, after reaction with antioxidants; therefore, this 7114



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Table 1. Analytical Performance of Various Antioxidant Compounds Calculated with Respect to the Voltammetric (DPV) and Spectrophotometric (Conventional CUPRAC) Methods polyphenol hydroxybenzoic acids

hydroxycinnamic acids

flavonols flavon-3-ols others

hydroxybenzoic acids

hydroxycinnamic acids

flavonols flavon-3-ols others

linear range (mM) vanillic acid (VA) gallic acid (GA) syringic acid (SA) p-coumaric acid (CMA) ferulic acid (FRA) chlorogenic acid (CGA) kaempferol (KAM) quercetin (QR) epicatechin (EC) catechin (CT) trolox (TR) L-ascorbic acid (AA) vanillic acid (VA) gallic acid (GA) syringic acid (SA) p-coumaric acid (CMA) ferulic acid (FRA) chlorogenic acid (CGA) kaempferol (KAM) quercetin (QR) epicatechin (EC) catechin (CT) trolox (TR) L-ascorbic acid (AA)

r

LOD (μM)

LOQ (μM)

62.69c + 0.032 141.41c + 0.037 69.44c + 0.036 60.77c + 0.084 54.23c + 0.006 126.68c + 0.068 74.11c + 0.024 193.77c + 0.051 145.93c − 0.039 134.71c + 0.045 46.66c + 0.019 51.32c − 0.035

0.9995 0.9998 0.9994 0.9998 0.9943 0.9988 0.9992 0.9995 0.9992 0.9953 0.9995 0.9990

0.23 0.12 0.23 0.62 0.05 0.24 0.15 0.12 0.12 0.15 0.18 0.31

0.76 0.39 0.78 2.07 0.16 0.80 0.48 0.39 0.40 0.50 0.61 1.02

23.05c 54.42c 24.83c 12.59c 23.39c 47.21c 34.15c 84.51c 60.38c 51.67c 15.36c 16.96c

0.9996 0.9999 0.9999 0.9989 0.9993 0.9995 0.9995 0.9992 0.9994 0.9993 0.9993 0.9996

0.41 0.28 0.24 1.82 0.13 0.38 0.45 0.15 0.13 0.36 0.29 0.58

1.36 0.94 0.80 6.07 0.45 1.27 1.49 0.50 0.42 1.19 0.98 1.94

linear calibration eq (10% EtOH)

Voltammetric Method (0.99−4.76) × 10−3 y= (0.49−2.38) × 10−3 y= (0.99−4.76) × 10−3 y= (0.99−4.76) × 10−3 y= (0.99−4.76) × 10−3 y= (0.49−2.38) × 10−3 y= (0.99−4.76) × 10−3 y= (0.49−2.38) × 10−3 y= (0.49−2.38) × 10−3 y= (0.49−2.38) × 10−3 y= (0.99−4.76) × 10−3 y= (0.99−4.76) × 10−3 y= Spectrophotometric Method (4.88−24.40) × 10−3 y= (2.44−12.20) × 10−3 y= (4.88−14.64) × 10−3 y= (12.20−61.00) × 10−3 y= (4.88−24.40) × 10−3 y= (4.88−24.40) × 10−3 y= (4.88−24.40) × 10−3 y= (2.44−12.20) × 10−3 y= (2.44−12.20) × 10−3 y= (4.88−24.40) × 10−3 y= (6.10−30.48) × 10−3 y= (6.10−30.48) × 10−3 y=

+ 0.021 + 0.034 + 0.013 + 0.051 + 0.007 + 0.024 + 0.034 + 0.028 + 0.017 + 0.041 − 0.010 − 0.022

Table 2. Trolox Equivalent Antioxidant Capacities (TEAC) of Various Antioxidant Compounds Determined According to DPV and Spectrophotometric (CUPRAC) Methodsa polyphenol trolox (TR) hydroxybenzoic acids vanillic acid (VA) gallic acid (GA) syringic acid (SA) hydroxycinnamic acids p-coumaric acid (CMA) ferulic acid (FRA) chlorogenic acid (CGA) flavonols kaempferol (KAM) quercetin (QR) flavon-3-ols epicatechin (EC) catechin (CT) others L-ascorbic acid (AA) a

TEACvoltammetric

TEACspectrophotometric

1.00

1.00

1.34 ± 0.01 3.03 ± 0.23 1.49 ± 0.08

1.50 ± 0.08 3.54 ± 0.05 1.61 ± 0.25

1.30 ± 0.08 1.16 ± 0.11 2.72 ± 0.19

0.82 ± 0.04 1.52 ± 0.09 3.07 ± 0.12

1.59 ± 0.12 4.15 ± 0.19

2.22 ± 0.15 5.50 ± 0.45

3.13 ± 0.25 2.89 ± 0.03

3.93 ± 0.17 3.36 ± 0.12

1.10 ± 0.07

1.10 ± 0.03

Figure 4. Correlation between spectrophotometric and voltammetric CUPRAC assays (r = 0.9791, N = 5) for found TEAC values of antioxidant standards.

containing Cu(Nc)22+ reagent caused a successive decrease of the DPV peak currents (Figure 5A). Apparently, the antioxidants present in herbal teas exerted an electrochemical effect similar to that of trolox, as represented by eq 1. The decrease in DPV current intensities of Cu(Nc)22+ reagent as a result of increasing herbal tea extract volume ratio (Vherbal teas/ Vtotal, %) is shown for the tested herbal tea extracts in Figure 5B. The good linearity between these two parameters confirms that the voltammetric−CUPRAC method can be used for the determination of TAC of herbal teas and similar complex antioxidant samples. The TAC values of herbal teas were calculated with the use of eq 4, where ΔIp is the difference in current intensity, m is the mass of dry herbal tea sample, V is volume, and DF is dilution factor.

Data presented as mean ± (t0.05s/n1/2), N = 5.

correlation is highly satisfactory. The slight discrepancies of the spectrophotometric TEAC values obtained by the CUPRAC method shown in Table 2 from those available in the literature15 may have arisen from the differences in solvent composition (i.e., measurements in this work were performed in 10% EtOH-containing aqueous media). TAC Measurements of Herbal Teas. Addition of certain amounts of herbal tea extracts to the electrochemical cell 7115



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Figure 6. Total antioxidant capacities (mmol TE/g) of herbal teas (green tea, marjoram, sage, and alchemilla) determined by differential pulse voltammetric and spectrophotometric CUPRAC methods. Data are presented as mean ± (t0.05s/n1/2), N = 5. (P = 0.05, Fexptl =1.875, Fcrit(table) = 6.608, Fexptl < Fcrit(table).)

validated. The proposed voltammetric method correlated linearly with the spectrophotometric method (r = 0.9803, N = 5), and the TAC values found with the two methods for herbal teas were compatible by ±3.2%. Performance and Prospects of the DPV−CUPRAC Method. The developed DPV−CUPRAC method has been demonstrated for a variety of antioxidant compounds and validated against the well-documented spectrophotometric− CUPRAC procedure and can be an interesting alternative to spectrophotometric methods for determining the TAC of phenolic compounds in some herbal teas. The voltammetric method may be potentially useful for turbid and colored samples in which plant pigments may obscure the color of cuprous−neocuproine chromophore in spectrophotometry. The proposed method is easy and fast and showed better sensitivity (lower detection limits) than similar electrochemical assays. The electrochemical results showed that TACs of herbal teas were in the order green tea > sage > marjoram > alchemilla, in compliance with the results of the spectrophotometric−CUPRAC assay. A separate turbidity investigation was undertaken in which TACs of filtered and unfiltered orange and peach nectars were assayed by DPV−and spectrophotometric−CUPRAC methods. The DPV−CUPRAC assay results (for both filtered and unfiltered samples) were close or comparable to those (for filtered sample) found by the spectrophotometric−CUPRAC method (Table 3). However, the unfiltered nectars gave high CUPRAC absorbances and therefore yielded positive errors in spectrophotometry. All spectrophotometric measurements, by nature, require the elimination of particulate matter (by filtration) before absorbance measurement. As a result, the proposed electrochemical assay proved to be efficient for turbid

Figure 5. (A) Differential pulse voltammograms of Cu(Nc)22+ reagent showing the effect of increasing herbal tea extract volume parts; (B) dependence of ΔIp on the volume ratio (Vherbal teas/Vtotal, %) of green tea (1), marjoram (2), sage (3), and alchemilla (4) herbal teas. The calibration lines were found y = 14.276x − 0.022 r = 0.9997; y = 7.512x − 0.002, r = 0.9999; y = 6.825x + 0.023, r = 0.9990; and y = 2.231x + 0.018, r = 0.9995, respectively (N = 5). Supporting electrolyte was acetic acid/acetate buffer solution (0.1 M, pH 4.76), and other conditions were the same as in Figure 2.

TACvoltammetric (mmol/g) ΔΙ p ± intercept (mL) V V × total × extract × DF = (ΔΙ p/c)TR m (g) Vsample × 10−3

(4)

The analytical results for total antioxidant capacity (expressed in mmol trolox equivalents per g of dry herbal tea sample) by voltammetric method were found to be 1.23 ± 0.06, 0.80 ± 0.11, 0.68 ± 0.02, and 0.40 ± 0.03 mmol/g for green tea, sage, marjoram, and alchemilla, respectively. The corresponding spectrophotometric findings with the conventional CUPRAC method were 1.19 ± 0.11, 0.70 ± 0.02, 0.66 ± 0.01, and 0.38 ± 0.02 mmol/g for green tea, marjoram, sage, and alchemilla, respectively. The TAC values of tested herbal tea samples measured by the developed voltammetric method were in good agreement with those of spectrophotometric−CUPRAC (Figure 6). The two-way analysis of variance (ANOVA) comparison by the aid of F test of the mean-squares of “between treatments” and of residuals13 for a number of real samples (herbal teas) enabled the conclusion that there was no significant difference between the population means for a given sample. In other words, calculated total antioxidant contents were alike at a 95% confidence level (Fexptl = 1.875, Fcrit(table) = 6.608, Fexptl < Fcrit(table) at P = 0.05). Thus, the proposed methodology was

Table 3. TACs of Some Nectars As Assayed by DPV − and Spectrophotometric− CUPRAC Methodsa sample orange nectar peach nectar

TACDPV−CUPRAC (mmol trolox equiv/L nectar)

TACspect−CUPRAC (mmol trolox equiv/L nectar)

1.96 ± 0.03 (2.04 ± 0.09)

1.82 ± 0.01 (4.5 ± 0.21)

1.57 ± 0.08 (1.53 ± 0.08)

1.51 ± 0.03 (3.5 ± 0.17)

Data presented as mean ± (t0.05s/n1/2), N = 5. TAC values in parentheses represent turbid sample results. a

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(14) Tütem, E.; Apak, R. Spectrophotometric determination of trace amounts of copper(I) and reducing agents with neocuproine in the presence of copper(II). Analyst 1991, 116, 89−94. (15) Apak, R.; Gücļ ü, K.; Ö zyürek, M.; Ç elik, S. E. Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay. Microchim. Acta 2008, 160, 413−419. (16) Ç elik, S. E.; Ö zyürek, M.; Gücļ ü, K.; Apak, R. Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection. Anal. Chim. Acta 2010, 674, 79−88. (17) Blasco, A. J.; Rogerio, M. C.; González, M. C.; Escarpa, A. Electrochemical index as a screening method to determine “total polyphenolics” in foods: a proposal. Anal. Chim. Acta 2005, 539, 237− 244.

samples for which the widely used spectrophotometric TAC assays are not accurately responsive.

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AUTHOR INFORMATION

Corresponding Author

*(R.A.) Phone: +90 212 4737070. Fax: +90 212 473 7180. Email: [email protected]. Funding

S.B. thanks Istanbul University Research Fund, Bilimsel Arastirma Projeleri (BAP) Yurutucu Sekreterligi, for the support given to his Ph.D. Thesis Project 29746 and to Istanbul University, Institute of Pure and Applied Sciences (I.U. Fen Bilimleri Enstitüsü), for the support given to his Ph.D. thesis work with the title “Development of a Novel Electrochemical Method for Determining the Total Antioxidant Capacity of Foodstuff”. We expresss our gratitude to T.R. Ministry of Development for the Advanced Research Project of Istanbul University (2011K120320). Notes

The authors declare no competing financial interest.

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REFERENCES

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A Novel Differential Pulse Voltammetric (DPV) Method for Measuring

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